Oncolytic viruses (OVs) are promising agents to combine with nanoparticle delivery approaches because of the capacity for self-replication of the virus. In systemic delivery, targeting with nanoparticles may focus the viral load to the primary tumor cells as well as metastatic tumors to insure a productive initial infection. A single viral particle delivered to a tumor cell can replicate to become thousands of viral particles and induce cell lysis with subsequent infection of additional tumor cells.
Oncolytic viruses can be directed at several mechanisms of action and exploit validated genetic pathways known to be deregulated in many cancers and are directly cytolytic. Cancer gene therapy holds great promise due to the approach which takes advantage of the virus' ability to replicate within cancer cells to levels that are many logs higher than the input dose, lyse the infected cell and subsequently spread to adjacent cells.
Adenoviruses are commonly used in gene therapy for cancer due to their ability to infect a broad range of cells. Recombinant adenoviruses are predominantly derived from adenovirus serotype 5 (Ad5). Clinical evidence of therapeutic activity has been demonstrated for oncolytic virus, ONYX-015. Following initial positive preclinical studies, phase I, II, and III clinical trials of ONYX-015 have been conducted in head and neck, gastrointestinal, ovarian, brain, pancreatic and breast cancer as well as oral dysplasia using local injections. In particular, the oncolytic virus TAV-255 has shown improved viral replication attenuation in normal cells while retaining cytolytic activity in tumor cells by taking advantage of defects in the p53-tumor suppressor pathway.
Despite these advantages, the utility of OVs for cancer therapy, including metastatic cancer, is limited by 1) the lack of expression of surface receptors (CAR) for the most common OVs in certain cancers, 2) rapid clearance by the reticuloendothelial (RE) system in the liver and 3) neutralization by antibodies.
Although high transduction is achieved for CAR positive and CAR deficient cells in positively charged polymer coated Ad, it has been shown that cationic liposomes and polymeric particles are readily taken up non-specifically by various cells.
Furthermore, cationic lipids such as N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE) and 3β-[N-(dimethylaminoethane)carbamoyl]cholesterol (DC-Chol) have been tested in clinical trials but the resultant biological (therapeutic) effects with these vesicles were at best marginal, and the formulations were hampered by toxicity.
Bilamellar cationic liposomes have shown to protect adenovectors from preexisting humoral immune responses. However, a high distribution in the lungs and liver after i.v injection was exhibited. Similarly, artificial envelopment of nonenveloped viruses showed an extended blood circulation times following i.v. administration and reduced vector immunogenicity. Although this platform allows for an extended circulation time in the bloodstream and a reduced immune response due to PEGylation, more than 70% of virus in the cationic DOTAP:DOPE:DSPE-PEG liposomes was cleared from the bloodstream and accumulated in the lungs, liver and spleen within 5 minutes.
Finally, a surface masking technique was developed. It is based on multivalent copolymers of poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) to ablate all pathways of receptor-mediated infection, combined with dose modulation to achieve partial saturation of nonspecific uptake pathways. Administration of elevated doses of the polymer-coated virus showed an increase in blood circulation time. However, it also showed saturation of phagocytic liver capture. Furthermore, differences in the circulation times between naked Ad and HPMA-Ad in the bloodstream were not distinguishable until they reached higher doses. Since most adults have neutralizing antibodies against Ad, attempts to increase exposure levels through the administration of high doses of Ad vectors can lead to severe liver damage and therefore, high doses should not be administered.
Despite these limitations, marked clinical responses have been observed in some patients following treatment by local as well as systemic delivery, indicating that effective approaches to maximize viral exposure to the tumor cells could enhance the effectiveness of oncolytic viruses as a therapeutic agent.
Although local, intratumoral administration of adenovirus (Ad) has produced marked antitumor effects in cancer gene therapy, there remains a need to develop an Ad vector system for systemic administration that can be used to treat both primary and metastatic tumors.
Several drawbacks are attributed to rapid clearance of the virus from circulation before it can reach its target site in a tumor or metastases. FIG. 1 depicts methods for biotechnology drugs comprising viral particles previously known in the art.
As shown in FIG. 1A, clearance from the bloodstream is mediated through neutralizing antibodies, inflammatory responses, as well as a nonspecific uptake by other tissues such as the lung, liver, spleen, and suboptimal viral escape from the vascular compartment. When viral particles, shown as spiked hexagons, are injected into a blood vessel, they are detected by patient's antibodies, shown as Y. After filtration by the liver, the concentration of viral particles in blood has decreased. Over 80% are accumulated in the liver 10 min after administration and only very few viral particles actually remain in circulation to reach the target tumor cells.
A range of methods have been designed to overcome these limitations. In general, encapsulation of a virus with a cationic liposome or coating the viral capsid with a cationic polymer has been employed due to the net negative charge of the viral capsid. For example, surface modification of adenovirus with an arginine-grafted bioreducible polymer has been developed to improve transduction efficiency and immunogenicity in cancer gene therapy as shown in FIG. 1B. A surface modification method of adenovirus with an arginine-grafted bioreducible polymer has been developed to improve transduction efficiency and immunogenicity in cancer gene therapy. However, the efficacy of viral particles encapsulated in cationic particles is limited by low cell and tissue specificity.
These modified viral particles are primarily taken up by Kupffer cells in the liver and non-specific cells in other organs before the viral particles can reach cancer cells.
Encapsulation of negatively charged adenovirus in cationic liposomes has been used in the field to overcome rapid clearance from the circulation to evade the immune barrier. However, despite the promising in vitro results, cationic liposomal encapsulation in vivo has been hindered by toxicities, low tissue specificity, and poor serum stability due to incompatibility with the abundance of negatively charged macromolecules present in the physiological environment.
Recent studies reported that anionic liposomes enhance transfection in CAR deficient cells. Zhong et al. demonstrated that adenovirus encapsulated in anionic liposomes using a calcium-induced phase change method was capable of protecting adenovirus from neutralization (Zhong et al. Mol. Pharmaceutics, 2011). Zhong et al. also showed that anionic liposomes enhance and prolong adenovirus-mediated gene expression in airway epithelia (Zhong et al., Int. Journal of Nanomedicine, 2011).